Squamous cell carcinoma initially occurring on the tongue dorsum: a case series report with molecular analysis

Background Squamous cell carcinoma (SCC) of the dorsum of the tongue is extremely rare, and it clinically resembles various benign lesions. Somatic mutations in TP53 and some driver genes were implicated in the development of SCC; however, the somatic genetic characteristics of dorsal tongue SCC remain unknown. With a detailed analysis of gene mutations in dorsal tongue SCC, we aimed to better understand its biology. Methods Four cases of SCC initially occurring on the tongue dorsum were evaluated for clinical and histological findings and immunohistochemical expression of p53 and p16. Gene mutations were analyzed using next-generation sequencing with a custom panel of driver genes. Results We retrospectively investigated 557 cases of tongue SCC, and only four cases of SCC initially occurred on the tongue dorsum. The four patients (cases 1–4) were one woman and three men with a mean age of 53.75 years (range: 15–74 years). Histological analysis revealed well-differentiated SCC. Through molecular analysis, we identified pathogenic somatic mutations, namely, TP53 p.C176F (c.527G > T) in case 3 and TP53 p.R282W (c.844 C > T) in case 4. No pathogenic variants were identified in the PI3K/AKT or RAS/RAF pathways. The p53 immunohistochemical examination revealed a wild-type expression pattern in cases 1–3 and strong expression in case 4. The results of p16 immunostaining were negative in all cases. Conclusions We described four previously unreported genetic characteristics of dorsal tongue SCC. Somatic TP53 mutations may contribute to the development of a subset of dorsal tongue SCC; however, more cases with genetic analysis need to be accumulated.

A detailed analysis of gene mutations in dorsal tongue SCC could enhance our understanding of its biology.Here, we performed clinical, pathological, and genetic analyses of a case series of dorsal tongue SCC.

Patient selection
We retrospectively investigated 557 cases of tongue SCC obtained from the pathology files of Okayama University Hospital, Osaka University Dental Hospital, Kagawa University Hospital, and Kagawa Prefectural Central Hospital.Among SCC cases occurring on the tongue dorsum, we excluded cases of non-primary SCC and simultaneous SCC at the tongue dorsum and other sites.The final diagnosis of dorsal tongue SCC was made by two pathologists (SO and KH).Formalin-fixed, paraffin-embedded (FFPE) tissues were retrieved for four patients with dorsal tongue SCC (cases 1-4).This study was approved by the Ethical Review Board of the Graduate School of Dentistry, Osaka University (No. R5-E11).

Histological and immunohistochemical examination
Resected tissue samples were fixed with 10% formalin, embedded in paraffin, cut into 4-µm-thick serial sections, and used for hematoxylin and eosin (H&E) and immunohistochemical staining.TNM classification was performed according to the 8th edition of the Union for International Cancer Control.Immunohistochemical staining with a primary antibody against p53 (clone-DO7) and p16 (clone-E6H4) was performed using a Roche Ventana BenchMark GX autostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer's instructions.

Clinical findings Case 1
A 63-year-old female patient complained of discomfort in the dorsum of the tongue.She had no history or family history of cancer.She was a smoker and did not consume alcohol.Intraoral examination revealed a well-circumscribed, elastic, soft mass measuring 13 × 9 mm in the midline to the right side of the tongue dorsum (Fig. 1a).
The patient underwent surgical resection under general anesthesia.The tumor grade was T1N0M0 (stage I).No signs of recurrence or metastasis were observed at the final follow-up 72 months later.

Case 2
A 74-year-old male patient was followed up for dorsal tongue oral leukoplakia (OL) for 10 years.He had a history of bladder and ureteral cancer (urothelial carcinoma) but no family history of cancer.He was a smoker and did not consume alcohol.An intraoral examination revealed an irregular, white, flat lesion on the dorsal surface of the tongue (Fig. 1c).The patient underwent surgical resection under general anesthesia.The tumor grade was T1N0M0 (stage I).No signs of recurrence or metastasis were observed at the final follow-up visit 60 months later.

Case 3
A 63-year-old male patient was followed-up for dorsal tongue OL for 2 years.He had a history of myelodysplastic syndrome (MDS).He had undergone hematopoietic stem cell transplantation (HSCT) for MDS 11 years prior and developed chronic graft-versus-host disease in the gastrointestinal tract one year after HSCT.He had no history of smoking or alcohol consumption.Intraoral examination revealed a white, slightly elevated lesion with an irregular surface measuring 17 × 9 mm at the midline of the dorsum of the tongue (Fig. 1e).The patient underwent surgical resection under general anesthesia.The tumor grade was T1N0M0 (stage I).No signs of recurrence or metastasis were observed at the last follow-up visit 13 months later.

Case 4
A 15-year-old boy was followed-up for dorsal tongue OL.The patient had a history of inherited bone marrow failure syndrome (IBMFS).He had no history of smoking or alcohol consumption.Intraoral examination revealed an erythematous, slightly elevated lesion measuring 30 × 15 mm in the midline to the left side of the tongue dorsum (Fig. 1g).The patient underwent surgical resection under general anesthesia.The tumor grade was T2N0M0 (stage II).SCC recurred 52 months after resection, and the patient died of immunodeficiency due to IBMFS 104 months after resection.

Histological findings
Histological examination revealed the proliferation of neoplastic squamous epithelial cells with various levels of cytological atypia (Fig. 1b, d, f, and h).The tumors exhibited irregular epithelial stratification and keratin pearl formation within the ridges.In cases 1, 2 & 4, tumors exhibiting nest or island structures infiltrated connective tissues, and we diagnosed well-differentiated SCC (Fig. 1b, d, and h).In case 3, the tumor showed a warty keratinized surface and pushing infiltration pattern with mild cytological atypia.The histological findings in case 3 were consistent with verrucous carcinoma (VC), which is a subtype of SCC (Fig. 1f ).Moreover, coexistence of foci of well-differentiated conventional SCC with VC was observed in the deep area (Fig. 1f, black box).The conventional SCC components showed irregular nests of cells with higher nuclear to cytoplasmic ratios and increased size of nucleoli (Fig. 1f, black box).The distance from the deepest point of invasion of the conventional SCC component to the nearest focus of VC was approximately 1000 μm.Thus, we finally diagnosed case 3 as VC with dysplasia or minimal invasion (VCDMI) [35].There were no findings of vascular or perineural invasion in the specimens of either of the four cases.In addition, there were no findings suggestive of amyloidosis, granular cell tumors, lichen planus, or chronic candidiasis.No SCC lesions were detected at locations other than the dorsum of the tongue.

Molecular genetic analysis and immunohistochemical findings
Genetic mutations were evaluated in all cases using NGS with a custom panel, as previously described [32].NGS  d, f and h; 200 μm in black boxes identified a TP53 p.C176F (c.527G > T) somatic mutation classified as pathogenic in case 3 and a TP53 p.R282W (c.844 C > T) somatic mutation classified as pathogenic in case 4 (Table 1).No pathogenic variants of PIK3CA, AKT1, PTEN, BRAF, MAP3K3, KRAS, NRAS, HRAS, or RASA1 were identified.Immunohistochemical examination of p53 revealed a wild-type staining pattern (negative to weakly positive) in cases 1-3 (Fig. 2a to c), and a strong staining pattern in basal and suprabasal layers in case 4 (Fig. 2d).The p16 immunostaining results were negative in all four cases.The results of the genetic and immunohistochemical analyses in previously reported cases and the present series are summarized in Table 1.

Discussion
We retrospectively investigated 557 cases of tongue SCC, and only four cases of SCC initially occurred on the tongue dorsum.Clinicopathological and genetic characteristics differ depending on the oral anatomical sites of OSCC [36], suggesting that dorsal tongue SCC may have characteristics different from those of SCC occurring at other sites.To the best of our knowledge, this study is the first to report a case series involving the clinical, pathological, and genetic analyses of dorsal tongue SCC.
Verrucous carcinoma is a well differentiated non-metastatic SCC subtype that may progress to invasive conventional SCC [1].Approximately 20% of VC cases of the oral cavity coexist with a conventional SCC component [42].Patel et al. categorized VC into three types: pure VC, VCDMI, and SCC arising in VC (SCC-VC) [35].Cases with a distance of less than 2 mm from the deepest point of invasion of the conventional SCC component to the nearest focus of VC are defined as VCDMI, and cases with a distance of 2 mm or more are defined as SCC-VC [35].In case 3 of the present study, the distance from the deepest point of invasion of the conventional SCC component to the nearest focus of VC was approximately 1000 μm.Thus, we finally diagnosed case 3 as VCDMI.Compared to SCC-VC, VC and VCDMI show good prognosis with a low recurrence rate, either locally or in regional lymph nodes [35].Consistently, case 3 showed no signs of recurrence or metastasis at the last follow-up visit 13 months later.Somatic mutations in TP53 and genes associated with the PI3K/AKT and RAS/RAF signaling pathways have potential roles as drivers of HNSCC and OSCC development [1,[22][23][24][25][26][27].Kobayashi et al. reported that the most frequently mutated gene among 284 HNSCC cases was TP53 (67%), followed by PIK3CA (8%), AKT1 (4%), and HRAS (3%) [26].Similarly, frequently mutated genes in OSCC were TP53 (65-66%), PIK3CA (16.8-20%),AKT1 (0-14%), and HRAS (9-9.3%)[23,25].Although no mutations in the PI3K/AKT and RAS/RAF signaling pathways were detected in the present study, we identified somatic TP53 mutations in 50% of the cases (2/4 cases; Table 1).The results of the genetic analysis in the present study are consistent with the distribution of somatic mutations in HNSCC and OSCC [1,23,25,26].TP53 is activated in response to DNA damage and is mutated in most common human malignancies, with various ranges depending on the stage and etiology of the tumors [43].Both TP53 p.C176F and p.R282W mutations are in the DNA-binding domain of p53 (Table 1) and have been reported as pathogenic and hotspot mutations in HNSCC [44].Two previous dorsal tongue SCC cases have been reported with p53 immunohistochemical staining; in one case, a germline TP53 mutation was identified (Table 1) [19,20].Combining these cases with those reported in the present study, the frequency of occurrence of TP53 mutations was 60% (3/5 cases) and the frequency of mutant p53 immunostaining pattern (null or positive) was 50% (3/6 cases; Table 1).These observations suggest that somatic TP53 mutations are involved in the development of a subset of SCC of the tongue dorsum.
A limitation of this study is its small sample size owing to the rarity of dorsal tongue SCC.Further studies are required to determine the genetic characteristics of dorsal tongue SCC and the association between gene mutations and SCC biology in a larger cohort of cases.

Conclusions
In the present study, we describe four cases of previously unreported genetic characteristics of dorsal tongue SCC.Our results suggest that somatic TP53 mutations may contribute to the development of a subset of dorsal tongue SCC; however, more cases with genetic analysis need to be accumulated.

Table 1
Summary of characteristics of dorsal tongue SCC in previous reported cases and the present series SCC, squamous cell carcinoma; IHC, immunohistochemical staining; M, male; F, female; NA, not available; WT, wild-type; MDS, myelodysplastic syndrome; IBMFS, inherited bone marrow failure syndrome